[Luke017]

Thanks!

Ah, that makes sense, thanks! I am clearly still thinking on too big a scale, I’m sure a few more months working on the sequencing aspect and I’ll get used to it.

[Luke017]

Tagmentation/Index PCR Volume Cutting

Hi All,

I hope you are all doing OK in these challenging times and managing to keep busy!

With the help of this thread I managed to carry out my first successful Smart Seq 2 run. I am now just going through my protocol and adding up costs etc and I noticed in the updated 2019 protocol the tagmentation and index PCR step uses 10 times less of the nextera kit reagents whilst the amount of cDNA recommended (50-150pg is pretty much the same) compared to original 2012 publication. I obviously trust this recommendation but I just wanted to check if anyone has experience with this? This would basically make the kit last 10x longer than the supplier recommendation which just seems too good to be true! Also sadly I would need to hand pipette these small volumes so not sure if this cut down recommendation is only for liquid handling robot platforms?

Any offers of advice/help would be once again greatly appreciated!

Luke

[Simone78]

Hi All,

I hope you are all doing OK in these challenging times and managing to keep busy!

With the help of this thread I managed to carry out my first successful Smart Seq 2 run. I am now just going through my protocol and adding up costs etc and I noticed in the updated 2019 protocol the tagmentation and index PCR step uses 10 times less of the nextera kit reagents whilst the amount of cDNA recommended (50-150pg is pretty much the same) compared to original 2012 publication. I obviously trust this recommendation but I just wanted to check if anyone has experience with this? This would basically make the kit last 10x longer than the supplier recommendation which just seems too good to be true! Also sadly I would need to hand pipette these small volumes so not sure if this cut down recommendation is only for liquid handling robot platforms?

Any offers of advice/help would be once again greatly appreciated!

Luke

Hi Luke,
that´s correct, it´s not a mistake. When starting from 50-150 pg cDNA you don´t need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE buffer. We then make "working dilution" plates with already unique i5/i7 combinations in each well. You can then pipette them with a multichannel but of course it would be easier with a robot.

However, the Illumina i5/i7 allows you only to pool 384 cells at the most (24 i7 x 16 i5). We now generated additional sets of adaptors and ordered from IDT. Then we do something similar as described above, with the exception that we now know the initial concentration (the 1:5 dilution is the result of "trial and error", there is nothing scientific behind this particular ratio).
We dilute all the adaptors to 10 uM and combine equal volumes of i5 and i7 in working dilution plates. Again, each well has a unique combination of i5/i7, each at 5 uM conc. We then use 1 ul of this mix for a 5 ul Nextera reaction (0.5 ul cDNA, 0.5 ul ATM, 1 ul TD, 0.5 ul NT, 1.5 ul NPM, 1 ul i5/i7). If you use the Nextera ones you would still use 1 ul of 1:5 diluted i5/i7.

I hope this helps!
Best,
Simone

[Luke017]

Hi Luke,
that´s correct, it´s not a mistake. When starting from 50-150 pg cDNA you don´t need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE buffer. We then make "working dilution" plates with already unique i5/i7 combinations in each well. You can then pipette them with a multichannel but of course it would be easier with a robot.

However, the Illumina i5/i7 allows you only to pool 384 cells at the most (24 i7 x 16 i5). We now generated additional sets of adaptors and ordered from IDT. Then we do something similar as described above, with the exception that we now know the initial concentration (the 1:5 dilution is the result of "trial and error", there is nothing scientific behind this particular ratio).
We dilute all the adaptors to 10 uM and combine equal volumes of i5 and i7 in working dilution plates. Again, each well has a unique combination of i5/i7, each at 5 uM conc. We then use 1 ul of this mix for a 5 ul Nextera reaction (0.5 ul cDNA, 0.5 ul ATM, 1 ul TD, 0.5 ul NT, 1.5 ul NPM, 1 ul i5/i7). If you use the Nextera ones you would still use 1 ul of 1:5 diluted i5/i7.

I hope this helps!
Best,
Simone

This is a massive help, thank you so much once again!

[Luke017]

Hi Luke,
that´s correct, it´s not a mistake. When starting from 50-150 pg cDNA you don´t need to use the standard amount of i5/i7. We generally dilute the adaptors 1:5 with water or low-EDTA TE buffer. We then make "working dilution" plates with already unique i5/i7 combinations in each well. You can then pipette them with a multichannel but of course it would be easier with a robot.

However, the Illumina i5/i7 allows you only to pool 384 cells at the most (24 i7 x 16 i5). We now generated additional sets of adaptors and ordered from IDT. Then we do something similar as described above, with the exception that we now know the initial concentration (the 1:5 dilution is the result of "trial and error", there is nothing scientific behind this particular ratio).
We dilute all the adaptors to 10 uM and combine equal volumes of i5 and i7 in working dilution plates. Again, each well has a unique combination of i5/i7, each at 5 uM conc. We then use 1 ul of this mix for a 5 ul Nextera reaction (0.5 ul cDNA, 0.5 ul ATM, 1 ul TD, 0.5 ul NT, 1.5 ul NPM, 1 ul i5/i7). If you use the Nextera ones you would still use 1 ul of 1:5 diluted i5/i7.

I hope this helps!
Best,
Simone

Ah, this is amazing, thanks again for all of your help.

[Luke017]

Adaptor sequencing issue

Hi all,

Apologies for my constant requirement for help (the downside of not having a core sequencing facility within my department). Following my first Smart Seq 2 test run which was largely successful the bioinformatician who is doing the analysis pointed out that a higher proportion than expected of my sequencing is adapter (Fast QC plot attached). I was carrying out 150bp paired end sequencing so obviously this resulted in quite a lot of wasted sequencing. I was wondering if anyone else has had a similar issue and suggestions on adaptions I could make to avoid this? I was rather surprised as my tapestation traces of my library following tagmentation and index addition PCR were largely of the expected size with few fragments <100 bp (traces attached).

Maybe this is a more general issue that I should post in a different thread but I thought I might first see if anyone had any advice from a Smart Seq 2 point of view.

Once again and hopefully for a final time any input/suggestions anyone has would be much appreciated.

Luke

2020-08-11 Luke Smart Seq 2 Opti plate 6 Post Tagment and Index PCR.pdf

[JonahN]

rRNA depletion solution

Hey,
I was just wondering if there is any way to make the rRNA depletion solution on our own?
Thanks
SJ

Yes, you can with a DSN that I developed for our company. I developed it as an alternative to the current DSN on the market (made in the USA), PureRec Duplex-Specific Nuclease that is more specific for digesting dsDNA and DNA in DNA-RNA hybrids. Please check out Zymo Research (E1020S, E1020-200, and E1020-1000). It is a fraction of the cost and recombinant so you can perform multiple experiments for rRNA depletions . It is available for purchase and also we have free samples available. Hope this helps!